Impact of template denaturation prior to whole genome amplification on gene detection in high GC-content species, Burkholderia mallei and B. pseudomallei.

OBJECTIVE: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM). RESULTS: Whole genome amplicons from 22 B. mallei (BM) and 37 B. pseudomallei (BP) isolates were tested for > 500 ARDs using ARDM v.3.1. ARDM detected the following Burkholderia spp.-derived genes, aac(6), blaBP/MBL-3, blaABPS, penA-BP, and qacE, in both BM and BP while blaBP/MBL-1, macB, blaOXA-42/43 and penA-BC were observed in BP only. The method of denaturing template for whole genome amplification greatly affected the numbers and types of genes detected by the ARDM. BlaTEM was detected in nearly a third of BM and BP amplicons derived from thermally, but not chemically denatured templates. BlaTEM results were confirmed by PCR, with 81% concordance between methods. Sequences from 414-nt PCR amplicons (13 preparations) were 100% identical to the Klebsiella pneumoniae reference gene. Although blaTEM sequences have been observed in B. glumae, B. cepacia, and other undefined Burkholderia strains, this is the first report of such sequences in BM/BP/B. thailandensis (BT) clade. These results highlight the importance of sample preparation in achieving adequate genome coverage in methods requiring untargeted amplification before analysis.

Prevalence of malaria resistance-associated mutations in Plasmodium falciparum circulating in 2017-2018, Bo, Sierra Leone.

Introduction: In spite of promising medical, sociological, and engineering strategies and interventions to reduce the burden of disease, malaria remains a source of significant morbidity and mortality, especially among children in sub-Saharan Africa. In particular, progress in the development and administration of chemotherapeutic agents is threatened by evolved resistance to most of the antimalarials currently in use, including artemisinins. Methods: This study analyzed the prevalence of mutations associated with antimalarial resistance in Plasmodium falciparum from 95 clinical samples collected from individuals with clinically confirmed malaria at a hospital in Bo, Sierra Leone between May 2017 and December 2018. The combination of polymerase chain reaction amplification and subsequent high throughput DNA sequencing was used to determine the presence of resistance-associated mutations in five P. falciparum genes - pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13. The geographic origin of parasites was assigned using mitochondrial sequences. Results: Relevant mutations were detected in the pfcrt (22%), pfmdr1 (>58%), pfdhfr (100%) and pfdhps (>80%) genes while no resistance-associated mutations were found in the pfkelch13 gene. The mitochondrial barcodes were consistent with a West African parasite origin with one exception indicating an isolate imported from East Africa. Discussion: Detection of the pfmdr1 NFSND haplotype in 50% of the samples indicated the increasing prevalence of strains with elevated tolerance to artemeter + lumefantrine (AL) threatening the combination currently used to treat uncomplicated malaria in Sierra Leone. The frequency of mutations linked to resistance to antifolates suggests widespread resistance to the drug combination used for intermittent preventive treatment during pregnancy.

Comparative analysis of stalked and acorn barnacle adhesive proteomes.

Barnacles interest the scientific community for multiple reasons: their unique evolutionary trajectory, vast diversity and economic impact-as a harvested food source and also as one of the most prolific macroscopic hard biofouling organisms. A common, yet novel, trait among barnacles is adhesion, which has enabled a sessile adult existence and global colonization of the oceans. Barnacle adhesive is primarily composed of proteins, but knowledge of how the adhesive proteome varies across the tree of life is unknown due to a lack of genomic information. Here, we supplement previous mass spectrometry analyses of barnacle adhesive with recently sequenced genomes to compare the adhesive proteomes of Pollicipes pollicipes (Pedunculata) and Amphibalanus amphitrite (Sessilia). Although both species contain the same broad protein categories, we detail differences that exist between these species. The barnacle-unique cement proteins show the greatest difference between species, although these differences are diminished when amino acid composition and glycosylation potential are considered. By performing an in-depth comparison of the adhesive proteomes of these distantly related barnacle species, we show their similarities and provide a roadmap for future studies examining sequence-specific differences to identify the proteins responsible for functional differences across the barnacle tree of life.

Comparison of capillary and venous blood for malaria detection using two PCR-based assays in febrile patients in Sierra Leone.

BACKGROUND: Rapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive. METHODS: Paired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay. RESULTS: No significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood. CONCLUSIONS: No difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.

Escherichia coli as a Potential Reservoir of Antimicrobial Resistance Genes on the Island of O'ahu.

The problem of antimicrobial-resistant bacteria has not been adequately explored in the tropical island environment. To date, there has not been a systematic investigation into the prevalence and distribution of antimicrobial resistance determinants in the Hawaiian Islands. Urinary isolates are the most common bacterial pathogens encountered in the clinical laboratory. Therefore, the antimicrobial resistance determinant profiles of these organisms can serve as a sentinel of the overall antimicrobial resistance situation in a localized patient population. In this study, 82 clinical isolates of Escherichia coli derived from 82 distinct patients were collected at a large medical center on the island of O'ahu. Each isolate was evaluated for the presence of antimicrobial resistance genes using a microarray-based approach. A total of 36 antimicrobial resistance genes covering 10 classes of antimicrobial compounds were identified. Most isolates were found to harbor between 3 and 5 antimicrobial resistance genes. Only a few isolates were found to harbor more than 12 genes. Significantly, a high rate of phenotypic resistance to one of the first-line treatments for uncomplicated urinary tract infection (sulfamethoxazole) was identified. This phenotype was correlated to the presence of sulfonamides and trimethoprim resistance determinants. Since E. coli is one of the most encountered pathogens in the hospital environment, the presence of clinically relevant resistance determinants in isolates of this organism from a clinical setting on O'ahu is a significant finding that warrants further investigation.

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone.

BACKGROUND: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone. METHODS: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species. RESULTS: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens. CONCLUSIONS: Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

Comparison of seven methods for DNA extraction from prosomata of the acorn barnacle, Amphibalanus amphitrite.

Next generation sequencing (NGS) technologies can provide an understanding of the molecular processes involved in marine fouling by Amphibalanus spp. barnacles. Here, seven methods for extracting DNA from A. amphitrite prosomata were assessed with respect to recovery, purity and size distribution. Methods incorporating organic extractions generally resulted in low recovery of fragmented DNA. The most promising method was the commercial E.Z.N.A. Blood DNA Mini kit, which provided tens of micrograms of DNA of sufficient molecular weight for use in long-read NGS library preparation. Other kits resulted in DNA preps suitable for short read length NGS platforms.

A comparison of methods for DNA preparation prior to microarray analysis.

Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase I-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91-100% sensitivity and 100% specificity in the tested strains, requiring half as many manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.

Characterization of longitudinal canal tissue in the acorn barnacle Amphibalanus amphitrite.

The morphology and composition of tissue located within parietal shell canals of the barnacle Amphibalanus amphitrite are described. Longitudinal canal tissue nearly spans the length of side shell plates, terminating near the leading edge of the specimen basis in proximity to female reproductive tissue located throughout the peripheral sub-mantle region, i.e. mantle parenchyma. Microscopic examination of stained longitudinal canal sections reveal the presence of cell nuclei as well as an abundance of micron-sized spheroids staining positive for basic residues and lipids. Spheroids with the same staining profile are present extensively in ovarioles, particularly within oocytes which are readily identifiable at various developmental stages. Mass spectrometry analysis of longitudinal canal tissue compared to tissue collected from the mantle parenchyma reveals a nearly 50% overlap of the protein profile with the greatest number of sequence matches to vitellogenin, a glycolipoprotein playing a key role in vitellogenesis-yolk formation in developing oocytes. The morphological similarity and proximity to female reproductive tissue, combined with mass spectrometry of the two tissues, provides compelling evidence that one of several possible functions of longitudinal canal tissue is supporting the female reproductive system of A. amphitrite, thus expanding the understanding of the growth and development of this sessile marine organism.

Seroprevalence of hepatitis B surface antigen (HBsAg) in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The aim of this study was to determine the prevalence of hepatitis B surface antigen (HBsAg) among febrile individuals tested at Mercy Hospital Research Laboratory (MHRL) in Bo, Sierra Leone. RESULTS: A total of 860 febrile individuals ages 5 years and older were tested by MHRL between July 2012 and June 2013 with a Standard Diagnostics Bioline HBsAg rapid diagnostic test. The overall HBsAg prevalence rate was 13.7%, including a rate of 15.5% among males and 12.6% among females. The HBsAg rate did not differ by child or adult age group (p > 0.5). The prevalence rate in Bo was similar to the 11-15% HBsAg prevalence rates reported in the past decade from other studies across West Africa. Scaling up the infant hepatitis B vaccination program in Sierra Leone will be important for reducing the future burden of disease and premature death attributable to chronic viral hepatitis B disease.

Prevalence of markers of HIV infection among febrile adults and children in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The goal of this study was to examine the prevalence of HIV among febrile patients seeking care in Mercy Hospital, Bo, Sierra Leone, in 2012-2013. RESULTS: A total of 1207 febrile persons were tested for HIV with Determine™ and SD Bioline rapid diagnostic tests kits that detect the presence of HIV antibodies and HIV p24 antigens. The overall prevalence of HIV among the tested patients was 8.9%, which is considerably higher than the < 2% prevalence of HIV reported previously in the general population. While these results are not sufficient to prove a causal relationship, the obtained data imply that HIV positive individuals may be more likely to suffer from febrile infectious diseases than individuals without HIV infection. Increasing the availability and use of HIV testing services will allow antiretroviral therapy to be accessed in a timely manner and improve health status among people living with HIV.

Surveillance of Vector-Borne Infections (Chikungunya, Dengue, and Malaria) in Bo, Sierra Leone, 2012-2013.

Malaria remains a significant cause of morbidity and mortality in West Africa, but the contribution of other vector-borne infections (VBIs) to the burden of disease has been understudied. We used rapid diagnostic tests (RDTs) for three VBIs to test blood samples from 1,795 febrile residents of Bo City, Sierra Leone, over a 1-year period in 2012-2013. In total, 24% of the tests were positive for malaria, fewer than 5% were positive for markers of dengue virus infection, and 39% were positive for IgM directed against chikungunya virus (CHIKV) or a related alphavirus. In total, more than half (55%) of these febrile individuals tested positive for at least one of the three VBIs, which highlights the very high burden of vector-borne diseases in this population. The prevalence of positives on the Chikungunya IgM and dengue tests did not vary significantly with age (P > 0.36), but higher rates of malaria were observed in children < 15 years of age (P < 0.001). Positive results on the Chikungunya IgM RDTs were moderately correlated with rainfall (r2 = 0.599). Based on the high prevalence of positive results on the Chikungunya IgM RDTs from individuals Bo and its environs, there is a need to examine whether an ecological shift toward a greater burden from CHIKV or related alphaviruses is occurring in other parts of Sierra Leone or the West African region.

Antimicrobial resistance of Klebsiella pneumoniae stool isolates circulating in Kenya.

We sought to determine the genetic and phenotypic antimicrobial resistance (AMR) profiles of commensal Klebsiella spp. circulating in Kenya by testing human stool isolates of 87 K. pneumoniae and three K. oxytoca collected at eight locations. Over one-third of the isolates were resistant to ≥3 categories of antimicrobials and were considered multidrug-resistant (MDR). We then compared the resistance phenotype to the presence/absence of 238 AMR genes determined by a broad-spectrum microarray and PCR. Forty-six genes/gene families were identified conferring resistance to ß-lactams (ampC/blaDHA, blaCMY/LAT, blaLEN-1, blaOKP-A/OKP-B1, blaOXA-1-like family, blaOXY-1, blaSHV, blaTEM, blaCTX-M-1 and blaCTX-M-2 families), aminoglycosides (aac(3)-III, aac(6)-Ib, aad(A1/A2), aad(A4), aph(AI), aph3/str(A), aph6/str(B), and rmtB), macrolides (mac(A), mac(B), mph(A)/mph(K)), tetracyclines (tet(A), tet(B), tet(D), tet(G)), ansamycins (arr), phenicols (catA1/cat4, floR, cmlA, cmr), fluoroquinolones (qnrS), quaternary amines (qacEΔ1), streptothricin (sat2), sulfonamides (sul1, sul2, sul3), and diaminopyrimidines (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA13/21/22/23 family, dfrA14, dfrA15, dfrA16, dfrA17). This is the first profile of genes conferring resistance to multiple categories of antimicrobial agents in western and central Kenya. The large number and wide variety of resistance genes detected suggest the presence of significant selective pressure. The presence of five or more resistance determinants in almost two-thirds of the isolates points to the need for more effective, targeted public health policies and infection control/prevention measures.

Effect of Linker Length on Cell Capture by Poly(ethylene glycol)-Immobilized Antimicrobial Peptides.

Development of antimicrobial peptide (AMP)-functionalized materials has renewed interest in using poly(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategies-such as changing tether length-may result in large differences in the surface properties of the immobilized AMPs that are not easily predictable.

Finished Genome Sequence of the Highly Multidrug-Resistant Human Urine Isolate Citrobacter freundii Strain SL151.

Citrobacter freundii is a Gram-negative opportunistic pathogen that is increasingly being recognized as a causative agent of hospital-acquired urinary tract infections and an important reservoir of antimicrobial resistance determinants. In this report, we describe the finished genome sequence of C. freundii strain SL151, a highly multidrug-resistant human urine isolate.

Prevalence of Quinolone Resistance in Enterobacteriaceae from Sierra Leone and the Detection of qnrB Pseudogenes and Modified LexA Binding Sites.

A collection of 74 Enterobacteriaceae isolates found in Bo, Sierra Leone, were tested for quinolone antibiotic susceptibility and resistance mechanisms. The majority of isolates (62%) were resistant to quinolones, and 61% harbored chromosomal gyrA and/or parC mutations. Plasmid-mediated quinolone resistance genes were ubiquitous, with qnrB and aac(6')-Ib-cr being the most prevalent. Mutated LexA binding sites were found in all qnrB1 genes, and truncated qnrB pseudogenes were found in the majority of Citrobacter isolates.

High prevalence of multidrug resistant Enterobacteriaceae isolated from outpatient urine samples but not the hospital environment in Bo, Sierra Leone.

BACKGROUND: The rising level of antimicrobial resistance among bacterial pathogens is one of the most significant public health problems globally. While the antibiotic resistance of clinically important bacteria is closely tracked in many developed countries, the types and levels of resistance and multidrug resistance (MDR) among pathogens currently circulating in most countries of sub-Saharan Africa are virtually unknown. METHODS: From December 2013 to April 2014, we collected 93 urine specimens from all outpatients showing symptoms of urinary tract infection (UTI) and 189 fomite swabs from a small hospital in Bo, Sierra Leone. Culture on chromogenic agar combined with biochemical and DNA sequence-based assays was used to detect and identify the bacterial isolates. Their antimicrobial susceptibilities were determined using a panel of 11 antibiotics or antibiotic combinations. RESULTS: The 70 Enterobacteriaceae urine isolates were identified as Citrobacter freundii (n = 22), Klebsiella pneumoniae (n = 15), Enterobacter cloacae (n = 15), Escherichia coli (n = 13), Enterobacter sp./Leclercia sp. (n = 4) and Escherichia hermannii (n = 1). Antimicrobial susceptibility testing demonstrated that 85.7 % of these isolates were MDR while 64.3 % produced an extended-spectrum ß-lactamase (ESBL). The most notable observations included widespread resistance to sulphonamides (91.4 %), chloramphenicol (72.9 %), gentamycin (72.9 %), ampicillin with sulbactam (51.4 %) and ciprofloxacin (47.1 %) with C. freundii exhibiting the highest and E. coli the lowest prevalence of multidrug resistance. The environmental cultures resulted in only five Enterobacteriaceae isolates out of 189 collected with lower overall antibiotic resistance. CONCLUSIONS: The surprisingly high proportion of C. freundii found in urine of patients with suspected UTI supports earlier findings of the growing role of this pathogen in UTIs in low-resource countries. The isolates of all analyzed species showed worryingly high levels of resistance to both first- and second-line antibiotics as well as a high frequency of MDR and ESBL phenotypes, which likely resulted from the lack of consistent antibiotic stewardship policies in Sierra Leone. Analysis of hospital environmental isolates however suggested that fomites in this naturally ventilated hospital were not a major reservoir for Enterobacteriaceae or antibiotic resistance determinants.

Evanescent wave fluorescence biosensors: Advances of the last decade.

Biosensor development has been a highly dynamic field of research and has progressed rapidly over the past two decades. The advances have accompanied the breakthroughs in molecular biology, nanomaterial sciences, and most importantly computers and electronics. The subfield of evanescent wave fluorescence biosensors has also matured dramatically during this time. Fundamentally, this review builds on our earlier 2005 review. While a brief mention of seminal early work will be included, this current review will focus on new technological developments as well as technology commercialized in just the last decade. Evanescent wave biosensors have found a wide array applications ranging from clinical diagnostics to biodefense to food testing; advances in those applications and more are described herein.